1. Field of the Invention
The present invention is directed to a purified enzyme and, more particularly, to a novel, purified, constitutive mammalian nitric oxide synthase (NOS) that utilizes both L-arginine and arginine-rich peptides, oligopeptides (e.g., bradykinin (BK)), and proteins as substrates.
2. Description of Related Art
Nitric oxide synthases (NOS, EC 1.14.23) are important enzymes, which convert L-arginine to L-citrulline and nitric oxide (NO). Nitric oxide is a very short-lived free radical, which is rapidly oxidized to nitrite (NO2) and nitrate (NO3) which are measured as the stable inactive end products of nitric oxide formation. The significance, however, lies in the fact that NO appears to play a pivotal role in a wide variety of physiological and pathological processes in mammals. These processes include vasodilation and regulation of normal vascular tone, inhibition of platelet aggregation, neuronal transmission, cytostasis, hypotension associated with endotoxic shock, inflammatory response-induced tissue injury, mutagenesis, and formation of carcinogenic N-nitrosamines (Nathan, FASEB J. 6:3051-3064 (1992); Kiechle et al., Am. J. Clin. Pathol. 100:567-575 (1993)). For example, it is well-known in the art to treat humans afflicted with angina distress and cardiovascular disease with nitroglycerin, which acts as a vasodilating agent. In the body, nitroglycerin is converted to nitric oxide (NO), which is the pharmacologically active metabolite. See, Palmer et al., Nature 333:664-666 (1988). Thus, evidence that NO mediates functions as diverse as those which occur in the brain, the endothelium and the blood, has led to intense study into the biological roles of NO and the various distinct members of the NOS family. (See, e.g., Marletta, J. Biol. Chem. 268:12231-12234 (1993); Knowles et al., Biochem. J. 298:249-258 (1994)).
It is well known by those skilled in the art that multiple isoforms of the NOS enzyme exist and that they are generally classified into two broad categories: 1) constitutive and 2) inducible. These classes of NOS enzymes vary considerably in their size, amino acid sequence, activity and regulation, and exhibit a number of substantial differences, indicating differences in their molecular structures. Increasingly diverse biological functions are being attributed to the NO formed by these three major known types of NOS (Nathan, FASEB J. 6:3051 (1992); Marletta, J. Biol. Chem. 268:12231 (1993); Knowles et al., Biochem. J. 298:249 (1994); Griffith et al., Annu. Rev. Physiol. 57:707 (1995)). For example, cells such as neurons and vascular endothelial cells contain constitutive NOS isotypes, while macrophages and vascular endothelial cells express an inducible NOS.
Several isoforms of NOS's from different mammalian tissues and cells have been purified and characterized; in brain (nNOS) by Bredt and Snyder, Proc. Natl. Acad. Sci. USA 87:682-685 (1990); in endothelial cells (eNOS) by Forstermann et al., Biochem. Pharmacol. 42:1849-1857 (1991); in macrophages (iNOS) by Hibbs et al., Science 235:473 (1987) and Stuehr et al., Proc. Natl. Acad. Sci. U.S.A. 88:7773-7777 (1991); in hepatocytes by Knowles et al., Biochem. J. 279:833-836 (1990); in vascular cells by Wood et al., Biochem. Biophys. Res. Comm. 170:80-88; in neutrophils by Yui et al., J. Biol. Chem. 266:12544-12547 (1991) and Yui et al., J. Biol. Chem. 266:3369-3371 (1991); and in other tissues (see, e.g., Hevel et al., J. Biol. Chem. 266:22789-22791 (1991); Mayer et al., FEBS Lett. 277:215-219 (1990); Schmidt et al., Proc. Natl. Acad. Sci. U.S.A. 88:365-369 (1991); Ohshima et al., Biochem. Biophys. Res. Commun. 183:238-244 (1992); Hiki et al., J. Biochem. 111:556-558 (1992); Evans et al., Proc. Natl. Acad. Sci. U.S.A. 89:5361-5365 (1992); Sherman et al., Biochemistry 32:11600-11605 (1993)). U.S. Pat. No. 5,268,465 claims a cDNA molecule encoding all or a portion of a mammalian calmodulin-dependent nitric oxide synthase (nNOS), comprising between 12 and 4,000 nucleotides. U.S. Pat. No. 5,468,630 claims an isolated nucleic acid molecule comprising the nucleic acid sequence encoding a human inducible nitric oxide synthase (iNOS) protein. U.S. Pat. No. 5,498,539 claims an isolated nucleic acid molecule comprising the nucleic acid sequence encoding a bovine endothelial nitric oxide synthase (eNOS) protein having amino acid or nucleic acid sequences set forth in the specification.
It is also known that small amounts of NO generated by a constitutive NOS appear to act as a messenger molecule by activating soluble guanylate cyclase and, thus, increasing intracellular guanosine, 3′,5′-cyclic monophosphate (cGMP) and the induction of biological responses that are dependent on cGMP as a secondary messenger. For example, through this mechanism, endothelial derived NO induces relaxation of vascular smooth muscle and is identified as endothelium derived relaxing factor (Palmer et al., Nature 327:524-526 (1987) and Ignarro et al., Proc. Natl. Acad. Sci. USA 84:9265-9269 (1987)). In addition, neuronal nitric oxide can act as a neuro-transmitter by activating guanylate cyclase with important functions in the central nervous system and autonomic nervous systems. Bredt and Snyder, Proc. Natl. Acad. Sci. USA 86:9030-9033 (1989) and Burnett et al., Science 257:401-403 (1992). Moreover, various purified NOS enzymes have been identified as hemeproteins (Stuehr et al., J. Biol. Chem. 267:20547-20550 (1992); White et al., Biochemistry 31:6627-6631 (1992); McMillan et al., Proc. Natl. Acad. Sci. USA 89:11141-11145 (1992)) and flavoproteins (Hevel et al., J. Biol. Chem. 266:22789-22791 (1991); Bredt et al., J. Biol. Chem. 267:10976-10981 (1992)).
Thus, the catalytic mechanisms of the NOS enzymes have also been the subject of great interest (see, e.g., Marletta, J. Biol. Chem. 268:12231-12234 (1993)). Stable isotope studies have shown when that when L-arginine is the substrate for the enzyme, NO derives from one of the two equivalent guanidino nitrogens on the arginine moiety (Ignarro et al., 1987; Palmer et al., 1988), and that di-oxygen is the source of the oxygen atoms incorporated into citrulline and NO (Kwon et al., J. Biol. Chem. 265:13442-13445 (1990); Leone et al., J. Biol. Chem. 266:23790-23795 (1991)). Moreover, NG-hydroxy-L-arginine has been demonstrated to be an oxidative intermediate in the catalytic process (Stuehr et al., J. Biol. Chem. 266:6259-6263 (1991); Wallace et al., Biochem. Biophys. Res. Commun. 176:528-534 (1991); Pufahl et al., Biochemistry 31:6822-6828 (1992); Klatt et al., J. Biol. Chem. 268:14781-14787 (1993)).
Co-factors involved in the conversion of L-arginine to L-citrulline and NO synthesis include tetrahydrobiopterin (BH4), flavin adenine nucleotide (FAD), the flavin-ribitol phosphate part of FAD (FMN) and the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH). In addition, when the NOS enzyme has been derived from brain or endothelial cells, calcium and calmodulin are also required. (Bredt et al., Nature 351:714-718 (1991); Lamas et al., Proc. Natl. Acad. Sci. U.S.A. 89:6348-6352 (1992); Lyons et al., J. Biol. Chem. 267:6370-6374 (1992); Lowenstein et al., Proc. Natl. Acad. Sci. U.S.A. 89:6711-6715 (1992); Xie et al., Science 256:225-228 (1992)). Calmodulin is a well-known protein binder for Ca2+, ubiquitously found in plant and animal cells. The Ca2+-calmodulin complex thus formed is known to bind to various target proteins in the cell, and thereby alter their activity.
Thus, it will be appreciated that nitric oxide has both normal physiologic intracellular and extracellular regulatory functions. However, excessive production of nitric oxide is detrimental. For example, when vascular endothelial cells are stimulated to express a NOS enzyme by a bacterial endotoxin, such as for example bacterial lipopolysaccharide (LPS), and inflammatory cytokines are elevated, the excess amounts of nitric oxides that are produced contribute to the vascular collapse seen in sepsis. Busse and Mulsch, FEBS Lett. 265:133-136 (1990). It is also known that when vascular cells are stimulated to express a NOS enzyme by inflammatory cytokines, the excess amounts of nitric oxides cause massive dilation of blood vessels and sustained hypotension commonly encountered in septic shock, and contribute to the eventual vascular collapse seen in sepsis. Id. It is also known that overproduction of nitric oxide in the lungs stimulated by immune complexes directly damages the lung. Mulligan et al., J. Immunol. 148:3086-3092 (1992). Induction of nitric oxide synthase in pancreatic islets impairs insulin secretion and contributes to the onset of juvenile diabetes. Corbett et al., J. Biol. Chem. 266:21351 (1991).
Thus, it will be appreciated that there is a great need in the medical community for the ability to control and regulate specific forms of NOS, particularly given its role in maintaining normal blood pressure and the devastating effect of excess NO on the cardiovascular, gastrointestinal and respiratory systems in humans. Thus considerable research has been expended to discover inhibitors and regulators of NOS activity. However, until the present invention, researchers have limited their work almost exclusively to the premise that L-arginine is the most physiologically relevant substrate of NOS. The studies have extended no further than the fact that dipeptides, such as Arg-Arg and Arg-Phe are also oxidized by crude NOS preparations derived from cultured endothelial cells and macrophages (Hecker et al., FEBS Lett. 294:221 (1991); Hecker et al., Proc. Natl. Acad. Sci. USA 87:8612 (1990); Hecker et al., J. Cardiovasc. Pharmacol. 20:S139 (1992)). Thus, although there have been many publications directed to analyses of arginine analogs or derivatives which inhibit NOS activity by blocking the use of arginine as a substrate for NO synthesis, or to the cofactors necessary for the conversion of arginine, no one until the present inventors has considered peptide, oligopeptide or protein substrates for the NOS enzyme. Hence, given the expectation that natural or synthetic arginine-rich peptide, oligopeptide or protein antagonists can function as NOS inhibitors, the present invention will greatly expand the number and types of inhibitors available for the regulation and control of NO production in the body. In addition, the use of a NOS to supply deficient individuals with NO-forming capability will be greatly enhanced by the purification of the nNOS-II class of enzymes. The present invention, therefore, will also provide many new ways to study the biological mechanisms involved in NO synthesis.